CN106198487A - The surface enhanced Raman detection method of Penicillin in Milk medicine - Google Patents
The surface enhanced Raman detection method of Penicillin in Milk medicine Download PDFInfo
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- CN106198487A CN106198487A CN201610523230.5A CN201610523230A CN106198487A CN 106198487 A CN106198487 A CN 106198487A CN 201610523230 A CN201610523230 A CN 201610523230A CN 106198487 A CN106198487 A CN 106198487A
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The surface enhanced Raman detection method of Penicillin in Milk medicine, the present invention relates to the detection method of Penicillin in Milk medicine.The present invention provides the surface enhanced Raman detection method of a kind of Penicillin in Milk medicine.This method: one, preparation has the Nano silver grain colloidal sol of SERS activity, then utilizes layer-by-layer to prepare Ag NPs assembling substrate on a glass substrate；Two, milk sample Organic substance to be measured/water mixed solvent is processed, obtain milk sample supernatant to be measured；Three, the glass substrate assembling Ag NPs being put in milk sample supernatant to be measured dipping, taking-up is dried, and obtains milk sample detection lug to be measured；Four, the surface enhanced raman spectroscopy of the milk sample detection lug to be measured obtained with Raman spectrometer testing procedure three, by the Raman collection of illustrative plates comparison of this spectrum Yu penicillin drug standards, it is achieved the qualitative detection to Penicillin in Milk medicine, qualitative detection limit as little as 10‑7mol/L。
Description
Technical field
The present invention relates to the detection method of Penicillin in Milk medicine.
Background technology
Penicillin belongs to beta-lactam antibiotic medicine, is to treat various bacterial infection diseases to use the most extensive and high
One of antibacterials of effect, use in terms of human diseases treatment and livestock breed aquatics in a large number.But, the abuse of antibiotic is existing
As the most generally existing, the abuse of penicillin can cause serious anaphylaxis and bacterial drug resistance, even causes the death of people.
In terms of livestock-raising, penicillin medicine is frequently used to the disease such as prevention and treatment bovine mastitis, therefore meeting in milk
There is Penicillin Residues, human health is constituted potentially hazardous.In order to ensure food safety, trace penicillin in conducting food
The detection research of drug molecule residual is the most necessary, the particularly detection of Penicillin in Milk drug molecule.Anticipate from tradition
Saying in justice, the analysis method of Penicillin in Milk drug residue mainly has high performance liquid chromatography, hexavalent chrome bio-removal and fluorescence
Polarization analysis etc..But these methods but having some limitations property.
" a kind of based on carboxylic peptide at the article of " analytical chemistry " (the Analytica Chmica Acta) of the 468th phase in 2002
Benzylpenicillin in the hexavalent chrome bio-removal detection by quantitative milk of inhibition of enzyme activity " (Biosensor analysis of
Penicillin G in milk based on the inhibition of carboxypeptidase activity) public
A kind of method having opened benzylpenicillin detected in milk.What the method utilized is the microbial receptor egg with carboxypeptidase activity
Bai Zuowei probe molecule, optionally the benzylpenicillin antibiolics of beta-lactam ring structure active, complete in detection milk
Thing.They use surfactant P-20 and amine coupling reagent kit that milk sample is purified process, wherein use polyclone
It is quantitative that antibody (R513) carries out selectivity as the specific recognition antibody of biosensor to the benzylpenicillin (antigen) in milk
Detection.By this technology, the detection limit of Penicillin in Milk G medicine can reach 2.6 μ g kg-1.Lacking of this detection method
Point is that not only biological sample preparation process is complicated and is difficult to preserve, and sample pre-treatments cost is high and detection overlong time.
" Food Chemistry " (the Food Chemistry) of the 141st phase in 2013 report a kind of based on high performance liquid chromatography-
Method (the An HPLC-DAD method of nine kinds of antibiotic medicines in diode array (HPLC-DAD) synchronous detecting ewe's milk
For the simultaneous determination of nine β-lactam antibiotics in ewe milk),
Including benzylpenicillin, ampicillin, cefalexin, cefazolin, cefoperazone, cloxacillin, dicloxacillin, oxazacillin and
Penicillin V.Wherein the extracting method of antibiotic remains is centrifugal by acetonitrile and Solid-Phase Extraction (SPE) and 0.45 μm microporous filter membrane
Filter process is purified process to the protein in ewe's milk sample.The quantitative limit (LOQs) of all these compounds all exists
3.4～8.6 μ g kg-1Scope.The shortcoming of this detection method is that experimental implementation process is complicated, and sample pretreatment process is time-consuming
And testing cost is high, greatly limit its application in actual analysis field.
The article of " Nai Ye association of the U.S. " (the American Dairy Science Association) of the 7th phase in 2014
" utilizing the research to Penicillin in Milk class drug degradation by-product of the Ultra Performance Liquid Chromatography-flight time mass spectrum multiple techniques "
(Research on degradation of penicillins in milk byβ-lactamase using ultra-
performance liquid chromatography coupled with time-of-flight mass
Spectromety), employing Ultra Performance Liquid Chromatography-flight time mass spectrum multiple techniques is disclosed in the presence of beta-lactamase
By the degradation by-products of research Penicillin in Milk class medicine (including benzylpenicillin, penicillin V and ampicillin) in turn
The method of the penicillin medicine contained in qualitative analysis milk.In the method, the pretreatment operation of mark-on milk sample is as follows:
By directly adding 10mL acetonitrile in 2mL mark-on milk sample: water (v/v, 3:1) mixed liquor, centrifugal under 3050 × g
10min, taking-up supernatant flows down to be dried at 40 DEG C of nitrogen and obtains solid residue, then is dissolved in by the residue obtained
In 2mL purified water and use the composite fibre resin microporous filter membrane of 0.45 and 0.22 μm to filter.Can enter after pretreatment
Row next step detection operation；Pretreatment operation is primarily to macromole interfering materials such as the protein removed in milk.This
The shortcoming of kind of detection method is not only can not carry out quantitative analysis, and complex operation step, analysis detection time length and one-tenth
This height.
The article of " Food Chemistry " (the Food Chemistry) of the 190th phase in 2016 " utilizes fluorescence polarization method to detect
The method of Penicillin in Milk G " (A novel fluorescence polarization assay for
Determination of penicillin G in milk) report one based on benzylpenicillin and suitable orchil conjugation
The Fluorescence Polarization assay of trace benzylpenicillin in penicillin antibody (biosensor) the detection by quantitative milk that labelling produces.He
Utilize purification kit that milk sample carries out pretreatment, use goat-anti rabbit polyclonal antibody (secondary antibodies) as in milk
The specific recognition antibody of benzylpenicillin medicine (antigen).The fluorescence polarization assay method that the document is proposed is used directly for
The detection of Penicillin in Milk G and do not disturbed by other materials in milk.By this method, its detection limit can reach
1nmol/L, this is fewer than maximum residue limit 12.0nmol/L that European Union (EU) specifies.This detection method utilizes purification kit
Carry out pre-treatment, use the goat-anti rabbit polyclonal antibody (secondary antibodies) of horseradish peroxidase labelling to build biosensor, real
Test cost to be greatly increased.
Up to the present, based on surface enhanced raman spectroscopy technology to grinding that Penicillin in Milk drug molecule detects
Study carefully that there is not been reported.
Summary of the invention
The present invention provides the surface enhanced Raman detection method of a kind of Penicillin in Milk medicine, overcomes lacking of prior art
Fall into, fill up the SERS technology blank in Penicillin in Milk drug molecule context of detection, for Penicillin in Milk class drug molecule
And the analysis detection of Beta-lactam medicine molecule provides new thinking.
The surface enhanced Raman detection method of the Penicillin in Milk medicine of the present invention, sequentially includes the following steps:
One, preparation has Nano silver grain (Ag NPs) colloidal sol of SERS activity, then utilizes layer-by-layer to exist
Ag NPs assembling substrate is prepared on glass substrate；
Two, add at Organic substance/water mixed solvent, supersound process 2～5min, then centrifugation in milk sample to be measured
Reason 10～20min, removal supernatant is at room temperature naturally evaporated to dryness and obtains residue；Deionized water is added again in residue
And supersound process 2～5min, then centrifuging treatment 5～10min, remove supernatant, successively with 0.45 μm and the water of 0.22 μm
Supernatant is filtered by mesentery, obtains milk sample supernatant to be measured；
Three, the glass substrate assembling Ag NPs is put in milk sample supernatant to be measured dipping, then by glass substrate
Take out, with deionized water rinsing and naturally dry, obtain milk sample detection lug to be measured；
Four, the surface enhanced raman spectroscopy of the milk sample detection lug to be measured obtained with Raman spectrometer testing procedure three,
Raman collection of illustrative plates comparison by this spectrum Yu penicillin drug standards, it is achieved to penicillin medicine qualitative in milk sample to be measured
Detection.
Milk sample to be measured is carried out pre-treatment by water secondary solvent two step isolation technics by the present invention, then with assembling Ag
NPs glass substrate impregnates, and the penicillin drug molecule absorption in milk sample to be measured is received at the noble silver with SERS activity
In rice corpuscles (Ag NPs) substrate, produce discernible raman spectral signal, by the body Raman light with penicillin standard substance
Spectrum comparison, if occurring the feature SERS signal peak of penicillin in milk sample, i.e. assert in milk sample have penicillin medicine
Exist, thus realize the qualitative of penicillin medicine and trace detection；This method is used to be possible not only to detect the green grass or young crops of Residues in Milk
Mycin medicine, by the spectrum comparison with standard substance, it is also possible to realize penicillin catabolite (such as penicilloic acid, penicillium sp
Acid etc.) carry out qualitative identification.
SERS technology is applied to the detection of Penicillin in Milk medicine by the present invention first, uses water secondary solvent two step to divide
Remove the most of interfering material in milk from technology, decrease the interfering component suppression (or sheltering) to SERS signal, improve
The sensitivity of SERS detection, qualitative detection limit can reach 10-7mol/L.Detection is without labelling, simple to operate；Detection is quick,
Needing sample size few and be easy to get, reagent water does not produce interference, is a kind of lossless detection method, this with low cost, environment is friendly
Well, SERS detection method is prone to commercial applications easily and fast.
Accompanying drawing explanation
Fig. 1 is that in test 1, benzylpenicillin concentration is 1 × 10-4The mark-on milk sample of mol/L and contrast test sample
SERS spectra and the Raman spectrogram of blank Ag NPs substrate；
Fig. 2 is that in test 1, benzylpenicillin concentration is 1 × 10-2The SERS spectra of the mark-on milk sample of mol/L and benzylpenicillin
Standard substance pressed powder and concentration are 1 × 10-1The Raman spectrogram of mol/L benzylpenicillin standard substance aqueous solution；
Fig. 3 is the SERS spectra of variable concentrations benzylpenicillin mark-on milk sample in test 1；
Fig. 4 is the benzylpenicillin concentration of mark-on milk sample and peak intensity standard curve in test 1；
Fig. 5 is SERS spectra and the benzylpenicillin standard substance solid of variable concentrations benzylpenicillin mark-on milk sample in test 2
The Raman spectrogram of powder.
Detailed description of the invention
Detailed description of the invention one: the surface enhanced Raman detection method of the Penicillin in Milk medicine of present embodiment, presses
Following steps are carried out:
One, preparation has Nano silver grain (Ag NPs) colloidal sol of SERS activity, then utilizes layer-by-layer to exist
Ag NPs assembling substrate is prepared on glass substrate；
Two, add at Organic substance/water mixed solvent, supersound process 2～5min, then centrifugation in milk sample to be measured
Reason 10～20min, removal supernatant is at room temperature naturally evaporated to dryness and obtains residue；Deionized water is added again in residue
And supersound process 2～5min, then centrifuging treatment 5～10min, remove supernatant, successively with 0.45 μm and the water of 0.22 μm
Supernatant is filtered by mesentery, obtains milk sample supernatant to be measured；
Three, the glass substrate assembling Ag NPs is put in milk sample supernatant to be measured dipping, then by glass substrate
Take out, with deionized water rinsing and naturally dry, obtain milk sample detection lug to be measured；
Four, the surface enhanced raman spectroscopy of the milk sample detection lug to be measured obtained with Raman spectrometer testing procedure three,
Raman collection of illustrative plates comparison by this spectrum Yu penicillin drug standards, it is achieved to penicillin medicine qualitative in milk sample to be measured
Detection.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: in step one, preparation has
Nano silver grain (Ag NPs) colloidal sol of SERS activity, then utilizes layer-by-layer to prepare Ag NPs on a glass substrate
Specifically comprising the following steps that of assembling substrate
A, by AgNO3Joining equipped with in the there-necked flask of deionized water, heating makes it dissolve under magnetic stirring；Continue
Heating, adding concentration when solution is warming up to the fluidized state of 98～100 DEG C is the sodium citrate aqueous solution of 1%, and stirring is formed
Vitreosol；Vitreosol keeps 50～60min under the slight boiling condition that temperature is 93～97 DEG C, then stops heating, natural
It is cooled to room temperature, obtains silver sol；Wherein AgNO3The ratio of quality and the volume of the sodium citrate aqueous solution that concentration is 1% be
9mg:1mL；
B, be firstly placed in the mixed solution of sulphuric acid and hydrogen peroxide boiling 1～2 hour by clean glass substrate, then spend from
Sub-water rinses, N2Air-blowing is done, and obtains hydroxylating glass substrate；Again this hydroxylating glass substrate being immersed in mass concentration is 0.3
～1% diallyl dimethyl amine hydrochlorate (PDDA) aqueous solution in keep 1～2h, rinse well with water after taking-up, and
Use N2Dry up, obtain the glass substrate of PDDA derivatization；Again the glass substrate of PDDA derivatization is impregnated in silver sol 4～
8h, taking-up water is rinsed well, and is used N2Dry up, obtain Ag NPs assembling substrate.Other is identical with detailed description of the invention one.
Detailed description of the invention three: Organic substance in present embodiment step 2 unlike detailed description of the invention one or two/
Water mixed solvent is the mixed liquor of dehydrated alcohol, chloroform and water；Or the mixed liquor of dehydrated alcohol, acetone and water；Other with
Detailed description of the invention one or two is identical.
Detailed description of the invention four: present embodiment is unlike detailed description of the invention three: the mixed liquor in step 2 is
Mixed for 7:2:3 by volume with water by dehydrated alcohol, chloroform；Or by dehydrated alcohol, acetone with water by volume
Mix for 7:3:4；Other is identical with detailed description of the invention three.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to four: in step 2 from
Heart separating treatment, centrifugal speed is 100～10000 revolutions per seconds；Other is identical with one of detailed description of the invention one to four.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: group in step 3
Dress Ag NPs glass substrate be placed in supernatant dipping time be 5～11 hours；Other is with detailed description of the invention one to five
One of identical.
Detailed description of the invention seven: present embodiment is unlike one of detailed description of the invention one to six: draw in step 4
Graceful spectrogrph is HORIBA LabRam ARAMIS type, and the wavelength of its excitation source is 633nm；Other is with detailed description of the invention one
Identical to one of six.
Detailed description of the invention eight: present embodiment is unlike one of detailed description of the invention one to seven: in step 4, draws
The time of graceful spectrometer collection signal is 3～5min；Other is identical with one of detailed description of the invention one to seven.
Detailed description of the invention nine: the penicillin that present embodiment is described unlike one of detailed description of the invention one to eight
For benzylpenicillin, penicillin K or penicillin V；Other is identical with one of detailed description of the invention one to eight.
The benzylpenicillin of present embodiment, penicillin K and penicillin V are all the typical penicillium sp including a beta-lactam nucleus
Element class antibiotic.
Verification experimental verification beneficial effects of the present invention with following:
Test 1: the surface enhanced Raman detection method of Penicillin in Milk medicine, sequentially includes the following steps:
One, 0.3564g benzylpenicillin (PEN) medicine it is directly appended in the milk without penicillin medicine and is settled to
10mL, it is thus achieved that concentration is 1 × 10-1The PEN mark-on milk mother solution of mol/L, then with the blank liquid milk without penicillin medicine
As solvent mark-on milk mother solution is diluted to respectively a series of desired concn: 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1 × 10-8Mol/L, obtains mark-on milk sample, the mark-on milk sample that will prepare
Store 7 days under the refrigerated condition of 4 degrees Celsius；The purpose of this operation is make testing sample closer to containing drug residue true
Real milk sample；
Two, preparation has Nano silver grain (Ag NPs) colloidal sol of SERS activity, then utilizes layer-by-layer to exist
Ag NPs assembling substrate is prepared on glass substrate；Concrete operating procedure is as follows: a, by 36mg AgNO3Join equipped with 200mL
In the there-necked flask of deionized water, heating makes it dissolve under magnetic stirring；When solution is heated to the fluidized state of 98 DEG C fast
Speed add 4mL concentration be the sodium citrate aqueous solution of 1%, in there-necked flask solution be gradually transformed into by water white transparency faint yellow,
Brown, ultimately generates the vitreosol of celadon；Vitreosol keeps 50min under the slight boiling condition of 95 DEG C, then stops adding
Heat, naturally cools to room temperature, obtains silver sol；B, the volume ratio that clean glass substrate is firstly placed on sulphuric acid and hydrogen peroxide are 7:3
Mixed solution in boil 1.5 hours, then with deionized water rinsing, N2Air-blowing is done, and obtains hydroxylating glass substrate；Again should
Hydroxylating glass substrate is immersed in diallyl dimethyl amine hydrochlorate (PDDA) aqueous solution that mass concentration is 0.5% guarantor
Hold 1h, rinse well with water after taking-up, and use N2Dry up, obtain the glass substrate of PDDA derivatization；Again by PDDA derivatization
Glass substrate impregnates 4h in silver sol, and taking-up water is rinsed well, and used N2Dry up, obtain Ag NPs assembling substrate；
Three, add in each mark-on milk sample to be measured by dehydrated alcohol, chloroform with water by volume for 7:2:3 mixes
The mixed solvent become, supersound process 2min promotes to dissolve, then centrifugation under conditions of centrifugal speed is 10000 revolutions per seconds
10min, removal supernatant is at room temperature naturally evaporated to dryness and obtains residue；Deionized water 4mL is added also again in residue
Supersound process 3min, then have Precipitation after centrifuging treatment 5min under conditions of centrifugal speed is 10000 revolutions per seconds, move
Go out supernatant, with the water system film of 0.45 μm and 0.22 μm, supernatant is filtered successively, obtain treating of different benzylpenicillin concentration
Survey mark-on milk sample supernatant；
Four, the glass substrate assembling Ag NPs is put into leaching in the mark-on milk sample supernatant of different benzylpenicillin concentration
Stain 8 hours, then takes out glass substrate, flushes three times with deionized water and naturally dry, and obtains a series of different benzylpenicillin
The mark-on milk sample detection lug of concentration；
Five, a series of mark-on milk samples obtained by HORIBA LabRam ARAMIS type Raman spectrometer acquisition step four
The spectroscopic data of product detection lug, the wavelength of excitation source is 633nm, and the signals collecting time is 5min, by this spectrum and benzylpenicillin
The Raman collection of illustrative plates comparison of drug standards, it is achieved to the qualitative detection of benzylpenicillin medicine in milk sample to be measured.
In this test, benzylpenicillin concentration is 1 × 10-4In the SERS spectra figure such as Fig. 1 of the mark-on milk sample of mol/L
Shown in curve a, the curve b in Fig. 1 is to be 1 by assemble Ag NPs glass substrate being impregnated into the concentration of unmixed solvent pre-treatment
×10-4Mol/L benzylpenicillin mark-on milk sample impregnates 8 hours, then glass substrate taking-up deionized water is flushed three times also
Naturally dry, the SERS spectra figure of the contrast test sample 1 obtained；C is the blank milk sample of blended solvent pre-treatment
SERS spectra；D is the Raman spectrum of Ag NPs substrate.It will be seen from figure 1 that without the 1 × 10 of pre-treatment-4Mol/L mark-on cattle
Milk sample does not produce Raman signal in Ag NPs substrate.Mark-on milk sample through mixed solvent pre-treatment then shows
The strongest Raman signal intensity and relatively low ambient interferences.Visible, volume ratio is that the mixed solvent of 7:2:3 is in mark-on milk
The Raman signal of benzylpenicillin strengthens tremendous contribution.
In this test, benzylpenicillin concentration is 1 × 10-2In the SERS spectra figure such as Fig. 2 of the mark-on milk sample of mol/L
Shown in curve c, a in Fig. 2 is the body Raman spectrum of benzylpenicillin standard substance pressed powder, the b in Fig. 2 be concentration be 1 ×
10-1The body Raman spectrum of mol/L benzylpenicillin standard substance aqueous solution.C from Fig. 2 is it can be seen that the feature of benzylpenicillin
SERS signal peak occurs in 1000cm-1Place, belongs to the eigen vibration of beta-lactam nucleus.By with benzylpenicillin pressed powder and
The body Raman spectrum contrast of aqueous solution, the benzylpenicillin can pointed out in mark-on milk sample assembles suprabasil at Ag NPs
SERS spectra, it was demonstrated that this test can the benzylpenicillin drug molecule of qualitative identification Residues in Milk；Penicillium sp thiazole can also be passed through
It is fatal that the spectrogram of acid standard substance judges whether the benzylpenicillin drug molecule remained there occurs that human body is had by decomposition reaction creating
The derivants such as the penicilloic acid of harm.
In this test, benzylpenicillin concentration is respectively 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1 × 10-8The SERS spectra of the mark-on milk sample of mol/L is as shown in Figure 3.Concentration a from top to bottom in Fig. 3
～h curve to be corresponding in turn to concentration be 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1
×10-8The sample of mol/L, from figure 3, it can be seen that along with the change of mark-on thing benzylpenicillin concentration, mark-on in milk testing sample
The Raman peaks intensity of thing changes the most therewith.Can be to cattle with the change of mark-on milk sample benzylpenicillin concentration by Raman peaks intensity
In milk sample, the content of benzylpenicillin carries out trace detection.Visible, this test can realize the benzylpenicillin medicine to Residues in Milk
Thing carries out trace SERS detection.By this method, the minimum detectability of Penicillin in Milk G residual reaches 1 × 10-7mol/L.Separately
Outward, at benzylpenicillin 1 × 10-3～1 × 10-7In mol/L concentration range, with Raman shift 1000cm-1The peak intensity at place is sat for vertical
Mark, maps for abscissa with mark-on Penicillin in Milk G concentration, can obtain standard curve as shown in Figure 4；Utilize standard curve
Can realize the detection by quantitative of benzylpenicillin concentration in milk sample to be measured.
Test 2: the surface enhanced Raman detection method of Penicillin in Milk medicine, sequentially includes the following steps:
One, 0.3564g benzylpenicillin (PEN) medicine it is directly appended in the milk without penicillin medicine and is settled to
10mL, it is thus achieved that concentration is 1 × 10-1The PEN mark-on milk mother solution of mol/L, then with the blank liquid milk without penicillin medicine
As solvent mark-on milk mother solution is diluted to respectively a series of desired concn: 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1 × 10-8Mol/L, obtains mark-on milk sample, the mark-on milk sample that will prepare
7 angel's testing samples are stored closer to the true milk containing drug residue under the refrigerated condition of 4 degrees Celsius；
Two, preparation has Nano silver grain (Ag NPs) colloidal sol of SERS activity, then utilizes layer-by-layer to exist
Ag NPs assembling substrate is prepared on glass substrate；Concrete operating procedure is as follows: a, by 36mg AgNO3Join equipped with 200mL
In the there-necked flask of deionized water, heating makes it dissolve under magnetic stirring；When solution is heated to the fluidized state of 98 DEG C fast
Speed add 4mL concentration be the sodium citrate aqueous solution of 1%, in there-necked flask solution be gradually transformed into by water white transparency faint yellow,
Brown, ultimately generates the vitreosol of celadon；Vitreosol keeps 50min under the slight boiling condition of 95 DEG C, then stops adding
Heat, naturally cools to room temperature, obtains silver sol；B, the volume ratio that clean glass substrate is firstly placed on sulphuric acid and hydrogen peroxide are 7:3
Mixed solution in boil 1.5 hours, then with deionized water rinsing, N2Air-blowing is done, and obtains hydroxylating glass substrate；Again should
Hydroxylating glass substrate is immersed in diallyl dimethyl amine hydrochlorate (PDDA) aqueous solution that mass concentration is 0.5% guarantor
Hold 1h, rinse well with water after taking-up, and use N2Dry up, obtain the glass substrate of PDDA derivatization；Again by PDDA derivatization
Glass substrate impregnates 4h in silver sol, and taking-up water is rinsed well, and used N2Dry up, obtain Ag NPs assembling substrate；
Three, add in each mark-on milk sample and mixed for 7:3:4 by volume with water by dehydrated alcohol, acetone
Mixed solvent, supersound process 3min promotes to dissolve, then centrifugation 10min under conditions of centrifugal speed is 8000 revolutions per seconds, moves
Go out supernatant to be at room temperature naturally evaporated to dryness and obtain residue；Deionized water 4mL supersound process is added again in residue
3min, then under conditions of centrifugal speed is 8000 revolutions per seconds, after centrifuging treatment 5min, have Precipitation, remove supernatant,
With the water system film of 0.45 μm and 0.22 μm, supernatant is filtered successively, obtain on a series of difference mark-on to be measured milk sample
Clear liquid；
Four, the glass substrate assembling Ag NPs is put into leaching in the mark-on milk sample supernatant of different benzylpenicillin concentration
Stain 10 hours, then takes out glass substrate, flushes three times with deionized water and naturally dry, obtaining a series of different mark-on
Milk sample detection lug；
Five, the mark-on milk sample detection obtained by HORIBA LabRam ARAMIS type Raman spectrometer acquisition step four
The spectroscopic data of sheet, the wavelength of excitation source is 633nm, and the signals collecting time is 5min；By this spectrum and benzylpenicillin medicine mark
The Raman collection of illustrative plates comparison of quasi-product, it is achieved to the qualitative detection of penicillin medicine in milk sample to be measured.
In this test, benzylpenicillin concentration is respectively 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1 × 10-8The SERS spectra of the mark-on milk sample of mol/L is as shown in a～h in Fig. 5, and in Fig. 5, I is penicillium sp
The body Raman spectrum of element G standard substance pressed powder.In Fig. 5, concentration a～h curve from top to bottom is corresponding in turn to concentration is 1
×10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7With 1 × 10-8The sample of mol/L, can see
Going out, the feature SERS signal peak of benzylpenicillin occurs in 1000cm-1Place, belongs to the eigen vibration of beta-lactam nucleus.By with green grass or young crops
The body Raman spectrum contrast of mycin G pressed powder, the benzylpenicillin can pointed out in mark-on milk sample assembles base at Ag NPs
SERS spectra at the end, it was demonstrated that this test can the existence of benzylpenicillin medicine of qualitative identification Residues in Milk, penicillium sp in milk
The minimum detectability of element G residual reaches 1 × 10-7mol/L.Patent of the present invention has obtained project of national nature science fund project
(21473078) subsidize.
Claims (9)
1. the surface enhanced Raman detection method of Penicillin in Milk medicine, it is characterised in that the method sequentially includes the following steps:
One, preparation has the Nano silver grain colloidal sol of SERS activity, then utilizes layer-by-layer to make on a glass substrate
Standby Ag NPs assembling substrate；
Two, in milk sample to be measured, Organic substance/water mixed solvent, supersound process 2～5min, then centrifuging treatment 10 are added
～20min, removal supernatant is at room temperature naturally evaporated to dryness and obtains residue；In residue, add deionized water again and surpass
Sonication 2～5min, then centrifuging treatment 5～10min, remove supernatant, successively with the water system film of 0.45 μm and 0.22 μm
Supernatant is filtered, obtains milk sample supernatant to be measured；
Three, the glass substrate assembling Ag NPs is put in milk sample supernatant to be measured dipping, then glass substrate is taken out,
With deionized water rinsing and naturally dry, obtain milk sample detection lug to be measured；
Four, the surface enhanced raman spectroscopy of the milk sample detection lug to be measured obtained with Raman spectrometer testing procedure three, should
Spectrum and the Raman collection of illustrative plates comparison of penicillin drug standards, it is achieved to the qualitative inspection of penicillin medicine in milk sample to be measured
Survey.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1, it is characterised in that step
In rapid one, preparation has the Nano silver grain colloidal sol of SERS activity, then utilizes layer-by-layer to prepare on a glass substrate
Specifically comprising the following steps that of Ag NPs assembling substrate
A, by AgNO3Joining equipped with in the there-necked flask of deionized water, heating makes it dissolve under magnetic stirring；Continue heating,
Adding concentration when solution is warming up to the fluidized state of 98～100 DEG C is the sodium citrate aqueous solution of 1%, and stirring is formed transparent molten
Glue；Vitreosol keeps 50～60min under the slight boiling condition that temperature is 93～97 DEG C, then stops heating, naturally cools to
Room temperature, obtains silver sol；Wherein AgNO3The ratio of quality and the volume of the sodium citrate aqueous solution that concentration is 1% be 9mg:
1mL；
B, it is firstly placed in the mixed solution of sulphuric acid and hydrogen peroxide boiling 1～2 hour by clean glass substrate, then uses deionized water
Flushing, N2Air-blowing is done, and obtains hydroxylating glass substrate；Again this hydroxylating glass substrate being immersed in mass concentration is 0.3～1%
Diallyl dimethyl amine hydrochlorate aqueous solution in keep 1～2h, rinse well with water after taking-up, and use N2Dry up,
Glass substrate to PDDA derivatization；Again the glass substrate of PDDA derivatization being impregnated in silver sol 4～8h, taking-up water rushes
Wash clean, and use N2Dry up, obtain Ag NPs assembling substrate.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
In step 2, Organic substance/water mixed solvent is the mixed liquor of dehydrated alcohol, chloroform and water；Or dehydrated alcohol, acetone and water
Mixed liquor.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 3, it is characterised in that step
Mixed liquor in rapid two is mixed for 7:2:3 with water by volume by dehydrated alcohol, chloroform.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
Centrifuging treatment in step 2, centrifugal speed is 100～10000 revolutions per seconds.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
In step 3 assemble Ag NPs glass substrate be placed in supernatant dipping time be 5～11 hours.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
In step 4, Raman spectrometer is HORIBA LabRam ARAMIS type, and the wavelength of its excitation source is 633nm.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
In step 4, the time of Raman spectrometer collection signal is 3～5min.
The surface enhanced Raman detection method of Penicillin in Milk medicine the most according to claim 1 and 2, it is characterised in that
Described penicillin is benzylpenicillin, penicillin K or penicillin V.
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